The unfolded protein response (UPR) is an essential cell signaling system that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER) and initiates a cellular response in order to maintain protein homeostasis. Aberrations in UPR signaling can lead to multitude of diseased states ranging from type 2 diabetes to neurological disorders. The most ancient branch of the UPR signaling pathways is initiated by activation of the ER-localized endoribonuclease (RNase) Ire1 upon ER stress. Once activated, Ire1 specifically cleaves the cytosolic mRNA of XBP1 to produce an active transcription factor that drives expression of UPR target genes to mitigate ER stress. In addition, Ire1 also promiscuously cleaves ER-localized mRNAs through the regulated Ire1-dependent decay (RIDD) pathway to reduce the burden of the incoming protein load. Although recent progress in understanding molecular basis of the Ire1/UPR pathway, fundamental mechanistic features of this pathway remain poorly understood. The goal of this application is to understand how Ire1 selectively finds and cleaves its mRNA substrates while avoiding non-specific cleavage of other cellular mRNAs. These studies will lead us to discover new steps in the UPR signaling, which can be targeted by drugs to treat diseases. The current research proposal is divided into two specific aims: 1. Determine how the RNase activity of Ire1 is regulated in cells. We have recently discovered that Ire1 is in a complex with the Sec61 translocon-ribosome in the ER membrane that facilitates cleavage of its correct mRNA substrates. We will first test the hypothesis that this complex may also prevent the non-specific RNase activity of Ire1 from accessing and cleaving cytosolic mRNAs by the use of RNA sequencing, biochemical and structural approaches. Second, we will establish a reconstituted proteoliposome system to test the role of the Sec61 translocon in regulating Ire1 oligomerization, phosphorylation and RNase activity in cells. 2. Determine how mRNA substrates are targeted to Ire1 in the ER membrane. We have shown that XBP1 mRNA, which encodes a soluble protein, utilizes the SRP pathway for targeting to the Ire1-Sec61 translocon complex for cleavage by Ire1. These and other studies raise two important questions. How are Ire1 substrate mRNAs that do not follow the SRP pathway targeted to Ire1 in the ER membrane? How Ire1 mRNA substrates are selectively targeted to Ire1-Sec61 translocon complexes in the ER membrane since Ire1 is a low abundant protein relative to Sec61? We will address these questions by a combination of ribosome profiling and biochemical reconstitution.